Molecular Ecology (2009) 18, 3443–3457 doi: 10.1111/j.1365-294X.2009.04292.x
Lineage diversification in a widespread species: roles forniche divergence and conservatism in the commonkingsnake, Lampropeltis getula
R. ALEXANDER PYRON*† and FRANK T. BURBRINK†
*Department of Biology, The Graduate School and University Center, The City University of New York, 365 5th Avenue NY,
NY 10016, USA, †Department of Biology, 6S-143, The College of Staten Island, 2800 Victory Blvd. Staten Island, NY 10314,
USA
Corresponde
E-mail: rpyro
� 2009 Black
Abstract
Niche conservatism and niche divergence are both important ecological mechanisms
associated with promoting allopatric speciation across geographical barriers. However,
the potential for variable responses in widely distributed organisms has not been fully
investigated. For allopatric sister lineages, three patterns for the interaction of ecological
niche preference and geographical barriers are possible: (i) niche conservatism at a
physical barrier; (ii) niche divergence at a physical barrier; and (iii) niche divergence in
the absence of a physical barrier. We test for the presence of these patterns in a
transcontinentally distributed snake species, the common kingsnake (Lampropeltisgetula), to determine the relative frequency of niche conservatism or divergence in a
single species complex inhabiting multiple distinct ecoregions. We infer the phyloge-
ographic structure of the kingsnake using a range-wide data set sampled for the
mitochondrial gene cytochrome b. We use coalescent simulation methods to test for the
presence of structured lineage formation vs. fragmentation of a widespread ancestor.
Finally, we use statistical techniques for creating and evaluating ecological niche models
to test for conservatism of ecological niche preferences. Significant geographical
structure is present in the kingsnake, for which coalescent tests indicate structured
population division. Surprisingly, we find evidence for all three patterns of conservatism
and divergence. This suggests that ecological niche preferences may be labile on recent
phylogenetic timescales, and that lineage formation in widespread species can result
from an interaction between inertial tendencies of niche conservatism and natural
selection on populations in ecologically divergent habitats.
Keywords: biogeography, genetic divergence, niche conservatism, niche modelling, speciation
Received 27 February 2009; revision received 12 May 2009; accepted 25 May 2009
Introduction
Speciation across geographical barriers can be influ-
enced by both niche conservatism (i.e. Peterson et al.
1999; Kozak & Wiens 2006) and niche divergence in
ecologically distinct habitats (i.e. Graham et al. 2004;
Raxworthy et al. 2007; Rissler & Apodaca 2007). The
idea that ancestral niches may be conserved across evo-
lutionary time (phylogenetic niche conservatism; Ricklefs
nce: R. Alexander Pyron, Fax: 718 982 3852;
well Publishing Ltd
& Latham 1992; Wiens 2004) has recently gained a great
deal of currency in the literature on speciation and the
study of broad scale patterns of lineage formation (Pet-
erson et al. 1999; Wiens & Graham 2005; Hawkins et al.
2006). Niche conservatism promotes allopatric diver-
gence in fragmented habitats by limiting adaptation to
new environments when populations maintain an
ancestral niche. Alternatively, niche divergence may
lead to lineage formation when populations adapt to
new environments (Wiens 2004; Wiens & Graham
2005). The mechanism by which this happens is well
defined, in terms of the fragmentation of a continuous
3444 R. A. PYRON and F. T . BURBRINK
habitat, which promotes geographical isolation, or dis-
persal into a new habitat, after which natural selection
subsequently promotes ecological differentiation (Futu-
yma 1998; Coyne & Orr 2004). However, the relative
importance of these processes for promoting lineage
formation in single widespread species complexes has
yet to be fully investigated. Whether both adaptive
divergence and ancestral conservatism of ecological
niche can influence lineage formation in widespread
taxa crossing multiple distinct ecoregions and putative
geographical barriers remains unknown.
At least three scenarios for allopatric speciation are
possible when considering the roles of geographic barri-
ers and ecological niche. The first is the presence of a
geographical barrier dividing ecologically distinct popu-
lations, which indicates niche divergence across the bar-
rier (e.g. Graham et al. 2004; Raxworthy et al. 2007).
The second scenario involves a barrier that divides eco-
logically similar populations, and indicates phylogenetic
niche conservatism (e.g. Kozak & Wiens 2006). The final
scenario is the presence of ecologically divergent popu-
lations on a continuous landscape without a physical
barrier separating them, indicating population diver-
gence promoted or at least reinforced by niche diver-
gence (e.g. Gee 2004). While these patterns have been
demonstrated on local geographic scales in various taxa,
it is unknown how broadly distributed organisms,
which inhabit a diversity of niches across their range,
respond to physical barriers and environmental varia-
tion, and whether one or a combination of the above
scenarios is the dominant mode of allopatric divergence
within in wide-ranging taxa.
Ectotherms such as reptiles are well suited for assess-
ing the impact of environment and geography on line-
age formation because of their low vagility and strong
responses to environmental factors (e.g. Howes et al.
2006; Burbrink et al. 2008; Fontanella et al. 2008). The
common kingsnake (Lampropeltis getula) is transconti-
nentally distributed in North America (NA; Conant &
Collins 1998; Krysko 2001) and also appears ideal for
investigating the roles of geographical barriers and eco-
logical niche on the formation of lineages because of its
ancient occupation of this region. Fossils have been
found in the central part of the U.S. dating to the late-
Miocene (Holman 2000) and by the Pliocene, the king-
snake had attained a distribution similar to the one
inhabited today (Holman 2000; Parmley & Walker
2003).
To examine phylogeographic structure in the North
American kingsnake, we assembled a range-wide
molecular data set of the mitochondrial gene cyto-
chrome b (cyt-b). While many issues regarding the use
of single mitochondrial gene estimates of phylogeny
have been raised (see Edwards & Beerli 2000), the use
of mtDNA for estimating phylogeographic structure
still has advantages (see Brito & Edwards 2008; Zink &
Barrowclough 2008), particularly for tracking recent
population divergence and associated ecological influ-
ences. While issues such as stochastic gene tree ⁄ species
tree discordance may influence phylogenetic estimates,
this does not directly affect the inference of local genetic
structure using mitochondrial loci.
First, we use phylogenetic reconstructions and diver-
gence time estimates to characterize the geographical
population structure in the kingsnake relative to the
presence of putative physical barriers in the range of
the organism. Second, given the possible uncertainty in
tree structure because of variance in the coalescent and
the ancient age by which the current range had been
attained (Pliocene), we test a scenario of structured
divergence across North America derived from our
maximum-likelihood tree of the real data vs. a null
model of an unstructured widespread ancestor. Reject-
ing the null model will permit us to determine which
clades can provide robust comparisons of ecological
niche characteristics.
Similar to Kozak & Wiens (2006, 2007), we use statis-
tical methods developed for assessing ecological niche
models (ENMs) to infer processes of niche conservatism
and niche divergence in the formation of lineages,
where empirical results in the kingsnake may exhibit
any or all of the following three patterns: (i) niche con-
servatism at a physical barrier; (ii) niche divergence at a
physical barrier; and (iii) niche divergence between
ecoregions that lack physical barriers. Using predicted
habitat suitability as a proxy for ecological niche, a lack
of statistically significant differences between ENMs for
lineages divided by geographical barriers would repre-
sent the signature of niche conservatism. Significant dif-
ferences in niche at a geographical barrier would
represent niche evolution or divergence in allopatry.
Significant niche differences in the absence of a physical
barrier would indicate that lineage formation was pro-
moted, or at least maintained, by niche divergence in
ecologically heterogeneous environments.
Methods and materials
Sequence acquisition
We obtained 201 tissue samples of Lampropeltis getula
taken throughout their known range and downloaded
60 gene sequences from a previous systematic study of
L. getula (Krysko & Judd 2006) from GenBank (File S1)
for a total of 261 samples (Fig. 1). For the tissue
samples, we used standard methods of proteinase K
digestion in lysis buffer followed by several rounds of
phenol ⁄ CHCl3 extraction (Sambrock & Russell 2001) or
� 2009 Blackwell Publishing Ltd
Fig. 1 Map of North America showing the sample localities, estimated range and identification legend for the major geographical
lineages identified in this study. Range limits adapted from Conant & Collins (1998), Stebbins (2003) and the results of the distribu-
tion modelling.
NI CHE EV OLUTION IN L A M PR O P E L TIS GE TU L A 3 44 5
Qiagen DNEasy kits (tissue protocol) to obtain total
genomic DNA from samples of shed skin, liver or mus-
cle tissue or whole blood. The complete mitochondrial
gene cyt-b was amplified using GoTaq Green Master
Mix (Promega Corp.) according to the manufacturer’s
specifications, with a 90-s extension time. The polymer-
ase chain reaction (PCR) products were cleaned using
1 lL of ExoSap-IT (USB Corp.) per 10 lL of PCR prod-
uct. The sequencing reaction consisted of 3 lL Beck-
man-Coulter DTCS, 2 lL primer (5 lm), 3 lL template
and 2 lL deionized water. Primers for the PCR and
cycle sequencing reactions were as follows; cyt-b ampli-
fication: H14910 and THRSN2 (Burbrink et al. 2000),
sequencing: LampSeq1F (5¢-GTA ATT ACA AAC CTA
CTA ACA GC-3¢) and LgetSeqRev2 (5¢-TTT GTT CCT
ART GGG TTR CTA GAG-3¢). For some particularly old
or degraded templates, cyt-b was amplified and
sequenced in two fragments, using H14910 + LgetSeq-
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Rev2 and LampSeq1F + THRSN2. Nucleotide sequences
were examined and aligned by eye using the program
SEQUENCHER 4.5 (Genecodes 2000). No sequences con-
tained any gaps or any stop codons that would have
suggested a pseudogene.
Phylogenetic inference
To test for the presence of population divergence and
assess the potential impact of geographical features on
lineage formation, we inferred the phylogeographic
structure of L. getula using Bayesian inference (BI) and
Maximum-Likelihood (ML) methods. For Bayesian phylo-
genetic inference, we used the program MRBAYES version
3.1.2 (Huelsenbeck & Ronquist 2001) to infer trees and
nodal support. To assess model complexity, we tested a
codon position partitioned General Time Reversible
model with gamma distributed rate heterogeneity and a
3446 R. A. PYRON and F. T . BURBRINK
proportion of invariant sites (3GTR + C + I) against a
codon position partitioned HKY85 + C + I model using
Posterior Bayes Factors (Kass & Raftery 1995), with PBF
>10 considered strong support for the more parameter-
ized model. Each analysis (two runs of four chains
each) was run for 2 · 107 generations. Convergence was
assessed using Gelman and Rubin’s r statistic (Gelman
et al. 1995). The lampropeltinine species Arizona elegans,
Lampropeltis calligaster, Lampropeltis triangulum and Lam-
propeltis extenuata were used as outgroups (File S1).
Maximum-Likelihood (ML) analysis was performed
using RAxMLv7.4.3 (Stamatakis 2006) with the same
data set used for the Bayesian analysis. A codon posi-
tion partitioned GTRMIX model was used, and 1000
nonparametric bootstrap replicates (Felsenstein 1985)
were performed to assess node support. Bayesian pos-
terior probabilities (Pp) greater than 95% are consid-
ered strong support for a clade, while bootstrap
proportions (BS) greater than 0.7 are considered strong
support (Hillis & Bull 1993; Felsenstein 2004).
Divergence time estimation
For divergence time estimation, we used relaxed-clock
phylogenetics methods (Drummond et al. 2006) in the
program BEAST version 1.4.8 (Drummond & Rambaut
2007). Divergence time estimation within species com-
plexes is hampered by the necessity for a compromise
between interspecific phylogenetic priors (tree priors)
and intraspecific coalescent priors. It is inappropriate to
combine inter- and intra-specific data in a single Bayes-
ian divergence time analysis, as no prior can adequately
account for both processes. We opt for the latter, com-
bining our ingroup data set with a single outgroup, the
sister taxon Lampropeltis extenuata (Pyron & Burbrink
2009a) and using coalescent priors for inferring diver-
gence times using the entire population level data set.
Although the inclusion of a nonconspecific outgroup
taxon does not render all of the assumptions of the
coalescent priors valid, this is a more conservative
mode of analysis than attempting to date a large
number of outgroups and a single representative from
each lineage using only one mitochondrial gene.
We tested both increasing (logistic and exponential
growth) and constant population size priors, which
yielded similar results; we report the data from the
constant population size prior. In addition, the inferred
estimates are concordant with a multi-gene phylogenetic
analysis of the tribe Lampropeltini (Pyron & Burbrink
2009a), suggesting that the estimated dates are robust to
variation in priors. An uncorrelated lognormal tree
prior, and lognormal fossil priors were used for diver-
gence time estimation under the relaxed-clock model in
BEAST (Drummond et al. 2006) using the same model
from the primary tree inference (3GTR + C + I). The
divergence between Lampropeltis extenuata and Lampro-
peltis getula was constrained to have occurred during the
Hemphillian, based on the fossils Stilosoma (Lampropeltis)
vetustum and Lampropeltis getula known from the middle
Hemphillian, late Miocene (Holman 2000). The mean of
the lognormal distribution was 6.875 Ma (1.9278) with a
standard deviation of 0.188, yielding a prior credible
interval of 4.75–9.94 Ma. No zero-offsets were used. The
analysis was run for 10 million generations, the first 2.5
million of which were discarded as burn-in. We
assumed convergence when the effective sample size of
the posterior probability distribution of all parameters
was >200 (Drummond et al. 2006), calculated in Tracer
v1.4 (Rambaut & Drummond 2007).
Historical biogeography
We used coalescent simulations in Mesquite version 2.5
(Maddison & Maddison 2008) to determine if the per-
ceived Structured Model from the ML tree fits the data
better than a widespread Fragmented Ancestor, based
on potential stochastic variance in the tree structure and
the observed number of deep coalescences (Knowles &
Maddison 2002; Fig. 2). The Fragmented Ancestor
model posits that all population divergences were in
effect concurrent and resulted from the fragmentation
of the widely distributed range of a common ancestor.
The presence of phylogeographic structure under this
model would be due to differential extinction of ances-
tral haplotypes among areas (Knowles 2001a, b;
Carstens et al. 2005). The Structured Model suggests
that a wide-ranging common ancestor orginating across
the Central US (Pyron & Burbrink 2009b) was first frag-
mented into two ancestral populations at the Missis-
sippi River and then each of those was subsequently
fragmented during colonization towards the western
and eastern US, respectively (Fig. 1).
For coalescent simulations, we first estimated Ne for L.
getula in each of the five geographically distinct areas
determined from phylogeographic analyses using values
for Q calculated in the program MIGRATE-N version 2.4
(Beerli 2008) under the following parameters: 15 short
chains for 200 000 generations and four long chains for
two million generations with four adaptive heating
chains, sampled every 20 generations following a burn-
in of 10 000 generations. Maximum-likelihood estimates
(MLE) were calculated three times to ensure conver-
gence upon similar values for Q. We converted Q to Ne
using the equation for maternally inherited mitochon-
drial DNA Q = Nel, where l = 3.0 · 10)8 site ⁄ genera-
tion calculated in BEAST version 1.4.8 and a generation
time of 3 years (Werler & Dixon 2000). We summed the
estimates of Ne for all areas to calculate Total Ne and
� 2009 Blackwell Publishing Ltd
Fig. 2 Illustration of the hypothetical
population ancestry models used for
coalescent simulations: (a) Structured
Model (the observed structure in the
ML tree in Fig. 3) vs. (b) Widespread
Ancestor (the fragmentation model).
Values on the trees represent propor-
tions of the total effective population
size (see text).
NI CHE EV OLUTION IN L A M PR O P E L TIS GE TU L A 3 44 7
scaled the branch widths of our hypothesized
population trees using the proportion of Total Ne that
each area comprised. Internal branches on the
Structured Model were scaled such that all branch
widths summed to Total Ne at any single point in time
(Carstens et al. 2004; Shepard & Burbrink 2008, 2009;
Fig. 2).
The method of counting the number of deep coales-
cences assumes that deep coalescent events are due to
incomplete lineage sorting and not migration among
populations (Knowles & Maddison 2002). In cases
where the number of deep coalescences may be inflated
by recent migration, it is important to account for
migration in simulations to build null models that
better reflect history under a given scenario (Shepard &
Burbrink 2009). Using the MLE of Total Ne, we simu-
lated 500 gene trees under a neutral coalescent process
with migration on the Fragmented Ancestor model at a
tree depth of 1 636 666 generations, which when based
on a 3-year generation time is equivalent to 4.91 Myr
(the approximate age estimated for the first divergence
within L. getula using the fossil calibrated relaxed-clock
phylogenetics method). To calculate the probability of
migration per individual per generation for these simu-
lations, we first multiplied values of M among adjacent
populations (areas) calculated in MIGRATE-N version 2.4
(Beerli 2008) by the Q of the receiving population to
derive the number of immigrants per generation among
pairs of adjacent populations. We divided these values
by the estimated Ne of the source population to calcu-
late the probability of emigration per individual per
generation in the source population, and then calcu-
lated the mean of all population pairs to derive the
average probability of migration per individual per
generation.
� 2009 Blackwell Publishing Ltd
We fit the simulated gene trees from the Fragmented
Ancestor model into the Structured Model, calculated
the number of deep gene coalescences (nDC) and built
a distribution of nDC values. We then fit our recon-
structed ML tree for L.getula to the Structured Model
and calculated the nDC value. If this observed nDC
falls below 95% of the distribution of nDC values calcu-
lated using the simulated gene trees (equivalent to one-
tailed P £ 0.05), then the Fragmented Ancestor model
will be rejected in favour of the Structured Model. To
calculate P values for the observed nDC values in these
analyses, we fit the distribution of simulated nDC val-
ues to a normal distribution with the given mean and
standard deviation (SD).
Niche modelling
Although the predicted habitat suitability from the
ENM results is not an absolute prediction of the true
fundamental or realized niche of an organism, it should
provide a reasonable proxy and allows for statistical
hypothesis testing regarding expressed niche prefer-
ences, at least with regard to the major environmental
conditions experienced by the organisms (Warren et al.
2008; review in Kozak et al. 2008). To assess the impact
of ecological niche on the formation and maintenance of
lineage separation, we modelled the predicted suitable
habitat of the inferred lineages of L. getula using maxi-
mum entropy methods (Elith et al. 2006; Phillips et al.
2006) in the program MAXENT version 3.2.19. The nine-
teen BIOCLIM variables from the WorldClim data set
(Hijmans et al. 2005) were used at 30-s spatial resolu-
tion (�1 km).
Many of the BIOCLIM variables are highly correlated,
and the relative contribution of each variable to the
3448 R. A. PYRON and F. T . BURBRINK
model for each lineage is uncertain. Thus, we followed
the protocol of Rissler & Apodaca (2007) and reduced
the data set to 11 biologically informative variables,
which are not significantly correlated across North
America: BIO1–3, 7–9, and 15–19. In addition, we used
the Level III Ecoregion designations for North America
provided by the U.S. EPA and Commission for Environ-
mental Cooperation. This layer, derived primarily from
Omernik (1987), classifies North America into 182 dis-
tinct ecoregions based on biological and environmental
ecosystem differentiation (Commission for Environmen-
tal Cooperation Working Group 1997). The ecoregions
were trimmed to the same extent as the BIOCLIM
variables and projected at 30-s spatial resolution.
To train the model, 733 georeferenced presence locali-
ties were obtained, comprising the samples used in our
phylogenetic analysis and additional georeferenced
museum records (File S2). The latter were obtained
either through the public web interface of museum col-
lections, or through the HerpNet database (http://herp-
net.org). Records with GPS coordinates were used as is;
all other records were georeferenced to the reported
locality using the description provided in the record.
Occurrences were assigned to lineages based on the cir-
cumscribed area as inferred from the primary phylogeo-
graphic analysis, and niche models were constructed
for each lineage.
We used auto features in Maxent (Phillips et al. 2006),
set the regularization multiplier to the default (1.0) and
allowed the algorithm to run to convergence (threshold
of 0.00001). The resulting niche predictions were pro-
jected onto a map of the U.S. in DIVA-GIS, with the
minimum training presence criteron used as the binary
threshold for predicted suitability. We attempted to
assess qualitatively the biological niche differentiation
between the lineages by determining which variables in
the model contributed the greatest proportion of
entropy to the model from the table given in the Maxent
output. Overlap in predicted suitable area was calcu-
lated between adjacent lineages by counting the number
of 30-s pixels predicted as suitable for both lineages.
This was converted to area by multiplying by 0.86 (30 s
of arc equals 0.93 km, thus a 30-s pixel equals 0.86 km2).
Niche differentiation
We assess differentiation in the predicted ENMs of the
lineages using the newly developed niche equivalency
methods of Warren et al. (2008). The program ENM-
Tools uses two niche similarity metrics, Schoener’s D
(Schoener 1968) and the newly developed ‘Warren
et al.’s’ I (Warren et al. 2008). These statistics quantify
predicted niche similarity, and range from 0 (no over-
lap) to 1 (identical niche models). We used the test of
niche equivalency in ENMTools, which evaluates equiv-
alency between ENMs by comparing the observed val-
ues of D and I for the two models with a distribution of
values of D and I based on randomized pseudorepli-
cates. This distribution is generated by randomly
assigning occurrence points from both groups into one
lineage or the other, simulating the potential overlap of
a group of points occurring across a given geographical
area (Warren et al. 2008). This allows for a one-tailed
test of dissimilarity from random for two ENMs. As we
are primarily reporting interactions between sister lin-
eages, we did not employ the possible phylogenetic cor-
rections for these analyses (Warren et al. 2008). We also
did not employ the more stringent tests for niche equiv-
alency given the available background, as no biological
justification for defining the available background is
evident for a single widespread species complex. We
calculated the observed D and I values and simulated
distributions of D and I using 100 pseudoreplicates for
all pairwise comparisons of the inferred lineages; only
those with relevance to our hypotheses are reported.
Because of computational constraints, the pseudorepli-
cates niche models were inferred using the 11 BIOCLIM
variables and the Level III Ecoregions projected at
2.5-min spatial resolution.
Results
The cyt-b gene sequenced for Lampropeltis getula and all
outgroup taxa measured 1117 bp with no indels or stop
codons in the reading frame for any sample. The
sequences have been deposited on GenBank under
the accession numbers FJ997648–FJ997848 (File S1). For
the BI phylogenetic analysis, we chose the 3GTR + C + I
model (PBF = 32.76). Gelman and Rubin’s r-statistic
(Gelman et al. 1995) was less than 1.001 for all parame-
ters after a burn-in of 5 · 106 generations (split standard
deviation among chains <0.01). Both analyses inferred
five major lineages with generally strong support for
both the lineages themselves and the relationships
between them (Figs 1 and 3). The primary geographical
lineages are as follows:
1 Eastern: A lineage comprising the kingsnakes of the
eastern seaboard of the United States, from New Jer-
sey to the Florida Keys and extending to the Apal-
achicola region in the Florida panhandle and
southeast Alabama.
2 Mississippi: This lineage ranges through the greater
Mississippi River drainage east of the Mississippi
River, from southern Illinois east to Ohio and western
West Virginia in the north, to the Tennessee and Ala-
bama river drainages of Georgia and Alabama in the
south.
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NI CHE EV OLUTION IN L A M PR O P E L TIS GE TU L A 3 44 9
3 Central: The Central lineage inhabits the Great Plains
and Mississippi River valley west of the Mississippi
River, from Iowa and Nebraska in the north to west-
central Texas and the western Gulf Slope in the
south, east to the Mississippi River.
4 Desert: The Desert lineage is found in the Chihuahan
Desert of west Texas, southern New Mexico (includ-
ing the Rio Grande River Valley), extreme southeast-
ern Arizona and eastern Mexico, along the Mexican
Plateau. May also occur in north central Arizona
(Fig. 4a).
5 Western: The Western lineage occurs west of the
Rocky Mountains, from the southern Great Basin in
Nevada and Utah, southern Oregon south to Baja
California, and most of Sonora, Mexico, east to south-
eastern Arizona.
All lineages were strongly supported by both posterior
probabilities (Pp>0.95) and bootstrap proportions
(BS > 0.9). The relationships among the lineages were
also all supported at greater than 95% Pp and 90% BS,
with the exception of the node subtending the Central
lineage and the Western and Desert lineages, which
received moderate support in the BI analysis (93% Pp)
and weak support in the ML analysis (59% BS). Lin-
eages of L. getula do not follow the currently designated
subspecies taxonomy and appear to correspond to his-
torical divergences at the Mississippi River (Western,
Desert & Central vs. Eastern & Mississippi); the Rocky
Mountains (Western & Desert vs. Central); the Cochise
Filter Barrier (Western vs. Desert); and the Appalachian
mountains ⁄ Chattahoochee River ⁄ Apalachicola River,
here termed the Apalachee formation for ease of refer-
ence (Eastern vs. Mississippi). While some lineages (e.g.
the Western and Eastern) exhibit strong concordance
between the geographical mtDNA lineage and the
currently described subspecies based on colour pattern
(see Fig. 3; Blanchard 1921, Blaney 1977), others (e.g.,
the Central and Gulf lineages) do not. Thus, while
morphological differentiation may be at least in part
related to ecological divergence, the strength and
underlying mechanisms of this pattern remain unclear.
Divergence dating and historical biogeography
Based on the prior distributions for the ages of the earli-
est known fossils of both taxa (Parmley & Holman
1995; Holman 2000), the dating analyses indicate that L.
getula diverged from its sister taxon Lampropeltis extenuata
during the Hemphillian of the late Miocene, �6.54 Ma
(95% Highest Posterior Density = 4.20–8.93 Ma). The
earliest divergence occurred at the Mississippi
River �4.91 Ma (95% HPD = 2.63–7.32 Ma) during the
late Hemphillian of the early Pliocene. This initial
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divergence at the Mississippi River and an area of
origin in the Central US corresponds with the area of
origin for the tribe Lampropeltini, of which L. getula is
a member (Pyron & Burbrink 2009a). Divergence
between the western (Western & Desert) lineages and
the Central lineage occurred during the Blancan of the
early Pliocene (4.06 Ma; 95% HPD = 1.86–6.12 Ma). As
fossils are known from Washington state dating to the
Pliocene (Parmley & Walker 2003), we infer that this
split occurred across the Rocky Mountains, and that the
Cochise Filter Barrier divergence represents a subse-
quent west-to-east vicariance event. Thus, the parapatry
of the Desert and Central lineages represents a zone of
secondary contact. The Cochise Filter Barrier (2.16 Ma;
95% HPD = 1.11–3.44 Ma) and Apalachee (1.94 Ma;
95% HPD = 0.75–3.35 Ma) divergences occurred at
approximately the same time at the Pliocene ⁄ Pleistocene
boundary.
In terms of ancestral population structure, the Struc-
tured Model posits an initial divergence at the Missis-
sippi River and subsequent divergences towards the
West Coast (Central and Desert + Western, then Desert
and Western clades) and the East Coast (Mississippi
and Eastern clades). This structured tree was then
tested against the Fragmented Ancestor model. Using
Migrate-N version 2.4 (Beerli 2008), we calculated a
maximum-likelihood estimate (MLE) of QTotal = 0.1034
(95% CI: 0.06–0.161). The MLE of QTotal equates to a
Total Ne of 3 446 666. Based on values of M and Q from
Migrate-N (Beerli 2008), we calculated a mean probabil-
ity of migration per individual per generation of
3.351 · 10)6, and used this value in coalescent simula-
tions. The number of deep coalescent events (nDC) for
our ML tree fit into the Structured Model was 54. This
value was significantly less than results from the coales-
cent simulations under the Fragmented Ancestor tested
against the Structured Model (mean nDC = 175.506,
SD = 20.81539, P = 2.586 · 10)9). Therefore, all subse-
quent tests of niche used the structure from the ML tree
representing the Structured model.
Niche modelling and equivalency
All predictions for each of the five lineages differed sig-
nificantly from random (binomial test, P < 0.00001) and
exhibited high sensitivity and specificity (AUC ⁄ ROC
values >0.95 for all lineages). The predicted distribu-
tions for each lineage closely matched their known
extent (Figs 1, 3 and 4). The use of museum records,
which precludes further discovery of between-lineage
dispersal, may cause an underprediction in niche over-
lap. However, overlap in predicted suitability is
observed between all adjacent lineages, and ranges from
0.0006% to 37% of the area of the lineages (Table 1;
Fig. 3 The ML tree produced using 261 samples of Lampropeltis getula and four outgroups under a 3 · GTRGAMMA model of evolu-
tion for the mitochondrial gene cytochrome b. Values above branches are the Posterior Probabilities from 1.5 · 107 post-burn-in sam-
ples; values below branches are the Bootstrap Proportion from 1000 nonparametric bootstrap replicates. Locations are given as
State ⁄ Country and County ⁄ Parish, followed by collection numbers as follows: FT Burbrink (FTB), KL Krysko (KLK), Museum of Ver-
tebrate Zoology (MVZ), University of Alabama Herpetological Collection (UAHC), Sternberg Museum (MHP), Yale Peabody
Museum (YPM), KM Enge (KME), University of Florida (UF), DB Means (DBM), TJ Hibbitts, Texas A&M University (TJH, MTH &
DL) Texas Natural History Collection (TNHC), Arkansas State University Museum of Vertebrate Zoology (ASUMZ), Illinois Natural
History Survey (INHS), Texas Cooperative Wildlife Collection (TCWC), Donald B. Shepard (DBS), KJ Irwin (KJI), LJ Vitt (LJV), Uni-
versity of Texas, Arlington (UTA), DG Mulcahy (DGM), San Diego Field Station (SD Field), University of Nevada, Reno (UNR), San
Diego Natural History Museum (SDSNH), California State University, Northridge (CSUN), Brad Moon, University of Louisiana,
Lafayette (CG). Specimens labelled KLK, KME, UF and DBM were obtained from GenBank, from Krysko & Judd (2006). Photographs
with voucher numbers are samples in the tree. Photographs for the Western and Central lineage are complements of SL & JT Collins,
CNAH.
3450 R. A. PYRON and F. T . BURBRINK
� 2009 Blackwell Publishing Ltd
Fig. 3 Continued
NI CHE EV OLUTION IN L A M PR O P E L TIS GE TU L A 3 45 1
Fig. 4). The variables with the greatest contribution to
the models for each lineage were as follows: Western
(BIO18: Precipitation of the Warmest Quarter; 44.7%),
Desert [BIO2: Mean Diurnal Range (Temperature);
35.1%], Central (BIO1: Annual Mean Temperature;
41.9%), Mississippi (BIO17: Precipitation of the Driest
Quarter; 62.6%) and Eastern (BIO7: Temperature
Annual Range: 23.1%).
� 2009 Blackwell Publishing Ltd
Schoener’s D and Warren et al.’s I values for the pair-
wise comparisons of interest were significantly lower
than expected from a random distribution (Table 1) for
all comparisons except for the Western and Desert lin-
eages, rejecting the null hypothesis of niche equivalency
for all adjacent lineages. The Western and Desert lineages
are not identical (D = 0.65, I = 0.523), but the interpre-
dicted suitability does not differ significantly from the
Fig. 4 Niche predictions for each inferred lineage at the points of primary geographical divergence and zones of secondary contact.
Overlap between predicted suitable habitat is indicated by the darkest intermediate shade.
Table 1 Area of predicted habitat suitability for the five inferred lineages of Lampropeltis getula and niche overlap calculations for
pairwise comparisons of adjacent clades
Lineage Area D (�X, SD, P) I (�X, SD, P)
Eastern 823 590 km2 — —
Mississippi 1 085 876 km2 — —
Central 1 892 157 km2 — —
Desert 726 695 km2 — —
Western 1 225 441 km2 — —
E vs. M 279 184 km2 0.265 (0.866, 0.031, <0.001) 0.523 (0.898, 0.022, <0.001)
C vs. M 304 064 km2 0.172 (0.811, 0.025, <0.001) 0.441 (0.860, 0.017, <0.001)
C vs. D 1139 km2 0.036 (0.312, 0.119, 0.01) 0.333 (0.542, 0.086, 0.008)
D vs. W 78 893 km2 0.120 (0.275, 0.149, 0.150) 0.428 (0.502, 0.100, 0.227)
Area calculated as the total number of 30 arc second pixels predicted as suitable using a binary threshold, multiplied by 0.86 (30 s of
arc = 0.86 km2). Niche overlap calculations for pairwise comparisons of adjacent clades. Overlap is the total area of the zone of
predicted overlap in environmental suitability from the ENMs. Values in parentheses give the mean and standard deviation of the
null distribution of D and I and the probability of the observed values from the niche equivalency test of Warren et al. (2008)
implemented in ENMTools.
3452 R. A. PYRON and F. T . BURBRINK
overlap between randomly assigned points. Given that
these lineages are allo- or peripatric (Figs 1, 3 and 4), this
is indicative of niche conservatism between these sister
lineages. At the zone of secondary contact between
the Desert and Central lineages, a small amount of in-
terpredicted suitability in the north appears to be
� 2009 Blackwell Publishing Ltd
NI CHE EV OLUTION IN L A M PR O P E L TIS GE TU L A 3 45 3
inhabited solely by the latter clade, whereas almost no
overlap occurs along the western Gulf slope in the
southern portion of the juncture, indicative of signifi-
cant niche divergence (Table 1).
Discussion
Historical biogeography of the common kingsnake
The common ancestor of the kingsnake originated in the
late Miocene and has since diverged to form five lineages
across major geographical barriers in North America. The
initial divergence at the Mississippi River (�5.0 Ma) is a
pattern, which has been documented in numerous species
of both plants and animals (Burbrink et al. 2000, 2008;
Howes et al. 2006; Soltis et al. 2006; Lemmon et al. 2007).
Subsequently, three lineages diverged westwards at
�4.0Ma(Central and Western ⁄ Desert) and 2.1Ma (Wes-
tern and Desert) and two eastwards�1.9 Ma (Eastern and
Mississippi). Divergences at the Cochise Filter Barrier
(�2.1 Ma) in the West and the Apalachee formation
(�1.9 Ma) in the East occurred more recently, and at a
similar time near the Pliocene ⁄ Pleistocene boundary. This
suggests that the congruent patterns of lineage divergence
observed at these features may be the result of similar
responses to physiographic and environmental shifts
during the late-Pliocene and early-Pleistocene (Soltis et al.
2006; Castoe et al. 2007).
At the Cochise Filter Barrier, the separation of the
Sonoran and Chihuahan desert provinces during the
late Pliocene (Morafka 1977) has been widely implicated
in the formation of geographical lineages across the
western deserts as refugia formed on either side of the
continental divide during the Pliocene and Pleistocene
(Jaeger et al. 2005; Smith & Farrell 2005; Devitt 2006;
Castoe et al. 2007; Mulcahy 2007). The observed
divergence in the Western and Desert lineages of the
kingsnake at �2.1 Ma is contemporaneous with the for-
mation of Sonoran ⁄ Chihuahan ⁄ Mojave clades in cactus
longhorn beetles (Moneleima armatum; Smith & Farrell
2005), western diamondback rattlesnakes (Crotalus atrox;
Castoe et al. 2007) and desert spiny lizards (Sceloporus
magister; Leache & Mulcahy 2007). Although few stud-
ies have dated divergences at the Apalachee formation,
most implicate the late-Pliocene and Pleistocene forma-
tion of glacial refugia in the southern Appalachians and
Florida in the separation of lineages (Burbrink et al.
2000; Soltis et al. 2006; Pauly et al. 2007).
All five lineages occupy geographically distinct habi-
tats according to niche modelling results and exhibit
varying areas of predicted overlap. The ENMs of the
sister Eastern and Mississippi lineages are found to be
significantly different based on randomization tests,
although the areas of predicted overlap are also associ-
� 2009 Blackwell Publishing Ltd
ated with apparent sympatry of the lineages (Figs 1, 3
and 4). Predicted areas of overlap between the sister
Western and Desert lineages are also associated with an
area of sympatry (Figs 1, 3 and 4), although the ENMs
for these lineages do not differ significantly, which
suggests a role for niche conservatism promoting speci-
ation across the Cochise Filter Barrier (Fig. 1). The
predicted overlap between the significantly different
ENMs of Central and Mississippi lineages is not asso-
ciated with widespread sympatry of the lineages,
suggesting that the Mississippi river is a strong barrier
to dispersal. The near-total lack of predicted overlap
between the Central and Desert lineages suggests a role
for niche divergence in promoting, or at least main-
taining, allopatric population segregation along an
environmental gradient.
Conservatism and divergence in ecological niche
Rather than finding that a single process of niche evolu-
tion played a dominant role, all three modes of niche-bar-
rier interaction are associated with lineage formation in
the kingsnake. We find (i) niche conservatism at the Co-
chise Filter Barrier; (ii) niche divergence at the Apalachee
Formation and the Mississippi River; and (iii) niche
divergence at the ecological transition between the adja-
cent Central and Desert ⁄ Western clades, which appears
to lack a physical barrier. The sister lineage pairs
(Western ⁄ Desert and Eastern ⁄ Mississippi) both appear to
have separated along axes of precipitation and tempera-
ture, although the particular aspects of those variables
are not consistent across the lineages. For a species occu-
pying a range the width of a continent, it is not surprising
to find a range of patterns suggesting different modes of
lineage divergence with respect to ecological niche. Niche
conservatism has been identified as the mechanism
responsible for the formation of many organisms such as
species of plethodontid salamanders in temperate areas
(Kozak & Wiens 2006; Shepard & Burbrink 2008, 2009). In
contrast, niche divergence has been found to promote
diversification in organisms such as tropical salamanders
occurring along elevational gradients (Kozak & Wiens
2007) and ecological gradients in other ectothermic verte-
brates (Graham et al. 2004; Raxworthy et al. 2007). How-
ever, to our knowledge, a role for both mechanisms in
diversification and lineage formation in a single species
has not been shown.
Although niche conservatism appears to be present
between some lineages in Lampropeltis getula, our
results show that ecological niches have not been
broadly conserved among lineages to a degree where
adjacent lineages share identical ENMs. This suggests
that environmental preferences are labile even on
recent timescales, and species may evolve significant
3454 R. A. PYRON and F. T . BURBRINK
differences even between recently diverged sister line-
age pairs as natural selection acts on populations in
ecologically heterogeneous habitats (Wiens 2004).
Indeed, ENM results predict almost complete ecologi-
cal separation between the peripatric Western and
Central lineages (Figs 1 and 4).
The indication from these results is that niche differ-
entiation is based on the dominant ecological feature of
the local environment. Thus, dry season precipitation
exerts the strongest influence on the Mississippi clade,
which inhabits the mesic Mississippi River drainage,
while annual temperature most strongly affects the Cen-
tral lineage, which occurs as far north as Nebraska, in
areas subject to extreme winters. However, even the
nondifferentiated Western and Desert lineages show
influence from different, presumably locally adapted
variables: daily temperature range for the Desert lineage
and rainy-season precipitation for the Western lineage.
Ultimately, while niche conservatism and divergence
may both influence lineage formation, the particular
niche differences between any given lineages may sim-
ply reflect historical contingencies rather than a unified
pattern of ecological influence.
Although niches must always be conserved in the
sense that descendant populations will inhabit similar
geographical areas or ecological niches as their immedi-
ate ancestors (i.e. Wiens & Graham 2005; Losos 2008a,
b; Wiens 2008), our results provide an important per-
spective on the influence of niche conservatism on spe-
ciation. The detection of niche equivalency between the
sister Western and Desert lineages, which are distrib-
uted across a putative climatic barrier, is indicative of
the classic scenario for speciation through niche conser-
vatism across a continuous landscape (Wiens 2004).
However, the ecological divergence between the
remaining lineages illustrates a more subtle point about
niche conservatism. The maintenance of lineages in geo-
graphically distinct areas must be due, at least in part,
to the conservation of niche preferences through natural
selection against individuals that disperse out of the
current niche (e.g. Holt & Gaines 1992; Wiens 2004).
Indeed, phylogenetic niche conservatism has been
shown to influence the continental distributions of
many organisms (Ricklefs & Latham 1992; Wiens et al.
2006, 2009; Pyron & Burbrink 2009b). While niche con-
servatism may exert a powerful influence on the distri-
bution of species, it is still possible for lineages to
exhibit divergence in environmental preferences on
short evolutionary timescales.
Conclusions
We find evidence that both niche conservatism and niche
divergence have played roles in promoting and main-
taining divergence between lineages of L. getula, as well
as zones of secondary contact between lineages. While
conservation of ancestral ecological conditions was
detected between some lineages, our results indicate that
ecological niche, or at least predicted habitat suitability
based on expressed environmental preferences, is labile
even on recent evolutionary timescales, and that niche
evolution and divergence may occur rapidly even
between sister lineages. Moreover, all of these processes
are shown to have occurred and are associated with
lineage formation in the common kingsnake. While
authors have recently argued about the basic defini-
tion of niche conservatism and the extent to which
niches are conserved (Losos 2008a, b), it is apparent
that it is not whether niches are conserved that is
important for phylogenetic studies, but the extent to
which niches are conserved and the impact that this
has on our understanding of the processes which influ-
ence speciation (Wiens & Graham 2005; Wiens 2008).
The evolutionary history of organisms, particularly of
recently diverged species complexes, is probably a mix-
ture of the inertial tendency of populations to maintain
their current niche and the action of natural selection on
populations, which differ in habitat across ecological
landscapes.
Acknowledgements
We would like to thank the following insitutions and persons
for providing samples or field assistance for this project: the
Museum of Vertebrate Zoology (JA McGuire), the University
of Texas, Arlington (JA Campbell, EN Smith, CJ Franklin and
R Jadin), the Texas Cooperative Wildlife Collection, Texas
A&M University (TJ Hibbitts), the University of Alabama (LJ
Rissler), the San Diego Natural History Museum (BD Hollings-
worth), the Peabody Museum, Yale University (G Watkins-Col-
well), California State University, Northridge and University of
Nevada, Reno (B Espinoza), the Florida Museum of Natural
History (KL Krysko), the Texas Natural History Collection, the
University of Texas, Austin (DC Cannatella, TJ LaDuc, D Hall),
Southeastern Louisiana Unviersity (BI Crother), the University
of Lousiana, Lafayette (B Moon), the Sternberg Museum, Fort
Hays State University (TW Taggart, CJ Schmidt, JT Collins),
the Illinois Natural History Survey (C Phillips), Karen Swaim
(Swaim Biological Inc.), the USGS San Diego Field Station, FM
Fontanella, P Warny, TJ Guiher, N Howe, SG Pyron, ME Py-
ron, DB Shepard, LJ Vitt, KJ Irwin, LK Irwin, CG Jimenez, SR
McNearney, RW Hansen, DG Mulcahy, C Roelke, A Harper,
PG Frank, C Whitney, GN Weatherman, S Ruane, MR Roch-
ford, RW Bryson, J Harrison, S Boback and D Siegel. Funding
for this research was provided in part by the Professional Staff
Congress-CUNY, the American Museum of Natural History
(Theodore Roosevelt Memorial Fund), the American Philosoph-
ical Society, the Center for North American Herpetology and
the College of Staten Island. We would also like to thank DB
Shepard for providing excellent comments that significantly
improved this manuscript.
� 2009 Blackwell Publishing Ltd
NI CHE EV OLUTION IN L A M PR O P E L TIS GE TU L A 3 45 5
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Supporting information
Additional supporting information may be found in the online
version of this article.
File S1 Collection localities for all specimens of Lampropeltis
getula used in this study, as well as GenBank accession
numbers for cytochrome b sequences for all samples.
File S2 733 georeferenced presence localities comprising the
samples used in our phylogenetic analysis and additional
georeferenced museum records.
Please note: Wiley-Blackwell are not responsible for the content
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authors. Any queries (other than missing material) should be
directed to the corresponding author for the article.